The beer, wine and baking yeast Saccharomyces cerevisiae has already been used for centuries for the production of bread, wine and beer owing to its characteristic of fermenting sugar to ethanol and carbon dioxide. In biotechnology, S. cerevisiae is used particularly in ethanol production for industrial purposes, in addition to the production of heterologous proteins. Ethanol is used in numerous branches of industry as an initial substrate for syntheses. Ethanol is gaining increasing importance as an alternative fuel, due to the increasingly scarce presence of oil, the rising oil prices and continuously increasing need for petrol worldwide.
In order to produce bioethanol inexpensively and efficiently, the use of lignocellulose-containing biomass, such as for example straw, waste from the timber industry and agriculture and the organic component of everyday household waste, presents itself as an initial substrate. Firstly, said biomass is very convenient and secondly is present in large quantities. The three major components of lignocellulose are lignin, cellulose and hemicellulose. Hemicellulose, which is the second most frequently occurring polymer after cellulose, is a highly branched heteropolymer. It consists of pentoses (L-arabinose, D-xylose), uronic acids (4-O-methyl-D-glucuronic acid, D-galacturonic acid) and hexoses (D-mannose, D-galactose, L-rhamnose, D-glucose) (see FIG. 1). Although, hemicellulose can be hydrolized more easily than cellulose, but it contains the pentoses L-arabinose and D-xylose, which can normally not be converted by the yeast S. cerevisae. 
In order to be able to use pentoses for fermentations, these must firstly enter the cell through the plasma membrane. Although S. cerevisiae is not able to metabolize D-xylose, it can uptake D-xylose into the cell. However, S. cerevisiae does not have a specific transporter. The transport takes place by means of the numerous hexosetransporters. The affinity of the transporters to D-xylose is, however, distinctly lower than to D-glucose (Kotter and Ciriacy, 1993). In yeasts which are able to metabolize D-xylose, such as for example P. stipitis, C. shehatae or P. tannophilus (Du Preez et al., 1986), there are both unspecific low-affinity transporters, which transport D-glucose, and also specific high-affinity proton symporters only for D-xylose (Hahn-Hagerdal et al., 2001).
In earlier experiments, some yeasts were found, such as for example Candida tropicalis, Pachysolen tannophilus, Pichia stipitis, Candida shehatae, which by nature ferment L-arabinose or can at least assimilate it. However, these yeast lack entirely the capability of fermenting L-arabinose to ethanol, or they only have a very low ethanol yield (Dien et al., 1996).
Conversion of L-Arabinose
In order for the pentose L-arabinose to be metabolised by S. cerevisiae, it must enter into the cell via transport proteins and be converted to the metabolite D-xylulose-5-phosphate in three enzymatic steps. These three enzymatic steps may be made available to the yeast by heterologously expressed genes. D-xylulose-5-phosphate functions as an intermediate of the pentose phosphate pathway and can be decomposed further to yield ethanol under anaerobic conditions in the cell (see FIG. 2).
Becker and Boles (2003) describe the engineering and the selection of a laboratory strain of S. cerevisiae which is able to use L-arabinose for growth and for fermenting it to ethanol. This was possible due to the over-expression of a bacterial L-arabinose metabolic pathway, consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous over-expression of yeast galactose permease transporting L-arabinose in the yeast strain.
Molecular analysis of the selected strain showed that the predetermining precondition for a use of L-arabinose is a lower activity of L-ribulokinase. However, inter alia, a very slow growth is reported from this yeast strain (see FIG. 2).
So far, it was only possible to express the native genes of bacterial arabinose metabolic pathways that are essential for metabolising arabinose in S. cerevisiae on single plasmids or to integrate them individually in the yeast genome, respectively (Karhumaa et al, 2006). This means that each yeast transformant with a functional arabinose metabolic pathway contained at least three plasmids or the genes integrated into the rDNA locus (Becker and Boles, 2003; Karhumaa et al, 2006).
The presence of the genes on different plasmids is associated with a number of disadvantages. On the one hand, plasmids that are present simultaneously represent additional stress for the yeast cells (“Plasmid stress”, Review of E. coli by Bailey (1993)). On the other hand, the plasmids used have strong homologies in their sequences, which can lead to loss of information within the plasmids due to homologous recombination (Wiedemann, 2005). However, the main disadvantages associated with the use of plasmids lie in the fact that they remain unstable in the strains without selection pressure and that they are not suitable for industrial use.
Moreover, it would be ideal for industrial applications if the microorganism used were able to metabolise all of the sugars present in the medium. Since the yeasts currently used industrially are not capable of metabolising the arabinose in the medium, it would be highly advantageous to provide the strains with this additional capablity in a stable manner.
The object of the present invention is therefore to provide means that overcome the disadvantages known from the prior art of introducing genes of a bacterial L-arabinose metabolic pathway into host cells individually, and which in particular may be usable for industrial yeast strains.
The object is solved according to the invention by the provision of nucleic acid molecules comprising three nucleic acid sequences, each of which codes for a polypeptide of an L-arabinose metabolic pathway, in particular a bacterial L-arabinose metabolic pathway.
A nucleic acid molecule according to the invention is a recombinant nucleic acid molecule. Furthermore, nucleic acid molecules according to the invention comprise dsDNA, ssDNA, PNA, CNA, RNA or mRNA, or combinations thereof.
The “L-arabinose metabolic pathway” or “bacterial L-arabinose metabolic pathway”, such as it occurs in E. coli, is shown in FIG. 2. This metabolic pathway contains 3 enzymes: L-arabinose isomerase, L-ribulokinase and L-ribulose-5-P-4-epimerase. The genes that code for these enzymes are called araA, araB and araD. L-arabinose isomerase converts L-arabinose to L-ribulose, which is further metabolised to L-ribulose-5-phosphate by the L-ribulokinase. Finally, the L-ribulose-5-P-4-epimerase converts L-ribulose-5-phosphate to D-xylulose-5-phosphate. The intermediate metabolite D-xylulose-5-phosphate is formed by the heterologously expressed genes of the L-arabinose metabolic pathway, particularly the bacterial L-arabinose metabolic pathway, in the yeast cell. D-xylulose-5-phosphate functions as an intermediate of the pentose phosphate pathway and can be further decomposed to ethanol under anaerobic conditions in a yeast cell. Enzymes of the xylose metabolic pathway are also found in fungi, and these and other enzymes isolated from eukaryotes can also be used as enzymes for the L-arabinose metabolic pathway.
The three nucleic acid sequences of the nucleic acid molecules according to the invention, each of which codes for a polypeptide of an L-arabinose metabolic pathway, are preferably araA (L-arabinose isomerase), araB (L-ribulokinase) and araD (L-ribulose-5-P-4-epimerase).
The nucleic acid molecules according to the invention preferably comprise nucleic acid sequences that are identical with the naturally occurring nucleic acid sequence or that have been codon-optimised for use in a host cell.
Each amino acid is encoded by one codon. However, there are several different codons that code for an individual amino acid. The genetic code is, thus, degenerated. The preferred codon selection for a corresponding amino acid varies from one organism to another. For example, problems may arise in heterologously expressed genes if the host organism or host cell has a very different codon usage. The gene can only be expressed very slowly, if at all. Differing codon usage may even be observed in genes of different metabolic pathways within the same organism. The glycolysis genes from S. cerevisiae are known to be expressed strongly. They have a highly restrictive codon usage. Adapting the codon usage of the bacterial genes of the arabinose metabolic pathway to the codon usage of the glycolysis genes from S. cerevisiae leads to improved arabinose metabolism in yeast.
For codon optimisation, the inventors did not rely on the usual platforms of synthetic gene designers for heterologous expression (such as Synthetic Gene Designer as described in Wu et al. 2006), instead they adapted the codon optimisation specifically to the codon usage of the glycolysis genes in the yeast. The glycolysis genes in the yeast have a highly restrictive codon usage, which is aligned with the frequency of the corresponding tRNA. The glycolysis genes use mainly codons for which there are high concentrations of the corresponding tRNAs, which in turn results in greater translation efficiency and gene expression (Bennetzen and Hall, 1982, Hoekema et al., 1987). In contrast, the usual synthetic gene designers are geared more to the average codon usage of all the genes in an organism, not just those that are highly expressed, and they also take into account other factors, such as stability. Accordingly, codon optimisation with the aid of such an electronic platform, such as the one described in Wu et al. 2006, results in a nucleic acid sequence that is entirely different from the one disclosed in this patent specification.
According to the invention, at least two of the three nucleic acid sequences, and preferably all three nucleic acid sequences, have been codon optimised for use in a host cell.
The nucleic acid sequence for araB (L-ribulokinase) and the nucleic acid sequence for araD (L-ribulose-5-P-4-epimerase) are preferably derived from E. coli. Thereby, the nucleic acid sequence for araB preferably comprises a nucleic acid sequence with SEQ ID NO: 1 and the nucleic acid sequence for araD preferably comprises a nucleic acid sequence with SEQ ID NO: 2.
The nucleic acid sequence with SEQ ID NO: 1 is the gene sequence of the open reading frame (ORF) of araBmut from E. coli in a codon-optimised form.
The nucleic acid sequence with SEQ ID NO: 2 is the gene sequence of the open reading frame (ORF) of araD from E. coli in a codon-optimised form.
The nucleic acid sequence for araA (L-arabinose isomerase) is preferably derived from Bacillus licheniformis or Clostridium acetobutylicum. 
These L-arabinose isomerases are advantageous for the growth of yeast transformants on an arabinose medium. Example 1 shows (see also FIG. 4) that, compared with the isomerase from B. subtilis, particularly the expression of the L-arabinose isomerase from C. acetobutylicum and B. licheniformis significantly improved the growth of yeast transformants on arabinose medium.
Thereby, the nucleic acid sequence for araA preferably comprises a nucleic acid sequence with SEQ ID NO: 3, 4 or 5.
The nucleic acid sequence with SEQ ID NO: 3 is the gene sequence of the open reading frame (ORF) of araA from Bacillus licheniformis in a codon-optimised form.
The nucleic acid sequence with SEQ ID NO: 4 is the gene sequence of the open reading frame (ORF) of araA from Bacillus licheniformis. 
The nucleic acid sequence with SEQ ID NO: 5 is the gene sequence of the open reading frame (ORF) of araA from Clostridium acetobutylicum. 
Accordingly, the nucleic acid sequences with SEQ ID NOs: 4 and 5 are naturally occurring nucleic acid sequences.
In a particularly preferred embodiment, a nucleic acid molecule according to the invention comprises the nucleic acid sequence with SEQ ID NO: 1, the nucleic acid sequence with SEQ ID NO: 2 and the nucleic acid sequence with SEQ ID NO: 3, 4 or 5. Most preferable is a nucleic acid molecule according to the invention that comprises the nucleic acid sequence with SEQ ID NO: 1, the nucleic acid sequence with SEQ ID NO: 2, and the nucleic acid sequence with SEQ ID NO: 3.
Yeast transformants that have the two codon-optimised genes of the kinase (araB, SEQ ID NO: 1) and the epimerase (araD, SEQ ID NO: 2), and yeast transformants in which all three genes have been codon-optimised (araB: SEQ ID NO: 1, araD: SEQ ID NO: 2 and araA: SEQ ID NO: 3), show a considerable growth advantage in a medium containing arabinose compared to yeast transformants that have only one codon-optimised gene. The strains show a considerably shorter lag phase and grow to their maximum optical density considerably faster (see example 2). The combination of three codon-optimised genes enables recombinant S. cerevisiae cells to convert L-arabinose considerably more efficiently.
The object is further solved according to the invention by the provision of expression cassettes comprising a nucleic acid molecule according to the invention.
Furthermore, the expression cassettes according to the invention preferably comprise promoter and terminator sequences.
Promoter sequences are preferably selected from HXT7, truncated HXT7, PFK1 FBA1, PGK1, ADH1 and TDH3.
Terminator sequences are preferably selected from CYC1, FBA1, PGK1, PFK1, ADH1 and TDH3.
Thereby, it is preferable that different pairs of promoter and terminator sequences control each of the three nucleic acid sequences. This is necessary to avoid possible homologous recombination between the promoter and/or terminator regions/sequences.
According to the invention, the pairs of promoter and terminator sequences are preferably selected from an HXT7 or truncated HXT7 promoter and CYC1 terminator, a PFK1 promoter and FBA1 terminator, and an FBA1 promoter and PGK1 terminator.
Particularly preferred is a nucleic acid sequence for araA controlled by the HXT7 or truncated HXT7 promoter and the CYC1 terminator.
Particularly preferred is a nucleic acid sequence for araB controlled by the PFK1 promoter and the FBA1 terminator.
Particularly preferred is a nucleic acid sequence for araD controlled by the FBA1 promoter and the PGK1 terminator.
For further details, see also example 3.
The expression cassettes according to the invention preferably comprise 5′ and/or 3′ recognition sequences as well.
Recognition sequences of the enzymes PacI and AscI are preferred.
The object is further solved according to the invention by provision of expression vectors, comprising a nucleic acid molecule or an expression cassette according to the invention.
The expression vectors according to the invention preferably comprise a selection marker as well.
The selection marker is preferably selected from a leucine marker, an uracil marker or a dominant antibiotic marker. A preferred dominant antibiotic marker is selected from geneticin, hygromycin and nourseothricin.
An expression vector according to the invention is preferably selected from the group p425H7synthAra, pRS303X, p3RS305X or p3RS306X.
For further details, see also example 3.
For industrial applications, it would be ideal if the microorganism used were capable of metabolising all of the sugars present in the medium. Since the yeasts that are currently used are not capable of metabolising the arabinose in the medium, it would be highly advantageous to provide the strains with this additional capablity in stable manner. In order to achieve this, an expression vector with genes of an arabinose metabolic pathway is highly beneficial. This expression vector can then be genomically integrated in a stable manner and can allow for the metabolisation of arabinose in industrial strains.
This invention succeeded (see also Examples) in constructing a vector that codes for an expression cassette with three genes of an arabinose metabolic pathway, particularly a bacterial metabolic pathway. In this way, it is possible to circumvent the problems that may arise when several plasmids are present in the same cell at the same time (“Plasmid stress”, Review of E. coli by Bailey (1993)). Furthermore, stable genomic integration of the arabinose metabolic pathway genes is enabled. The problems associated with constructing an expression cassette of the arabinose metabolic pathway genes and integrating it in a manner that is genomically stable have already been shown by Becker (2003) and Wiedemann (2005).
By selecting promoters and terminators in combination with using the improved L-arabinose isomerase and the codon-optimised versions of the genes involved, the construction of this functional expression cassette according to the invention was achieved.
The expression cassette constructed with the three genes according to the invention represents an excellent starting point for a direct genomic integration as well as enables subcloning into the integrative plasmids of the series pRS303X, pRS305X and pRS306X (Taxis and Knop, 2006).
Furthermore, a plurality of experimental obstacles and difficulties had to be overcome in the process of cloning the three genes with the different promoters and terminators, and these are reported in greater detail in the examples and figures.                Finding an L-arabinose isomerase that functions better, such as is more efficient, in yeast.        Cloning the isomerase proved to be difficult and time-consuming.        The vector according to the invention is the first vector described that contains all the essential genes for converting arabinose in yeast.        The vector contains all the genes in functional form and enables the recombinant yeast a good arabinose growth. Functionality as well as very good arabinose growth were by no means expected.        
The object is further solved according to the invention by providing host cells that contain a nucleic acid molecule according to the invention, an expression cassette according to the invention, or an expression vector according to the invention.
In a particularly preferred embodiment, a nucleic acid molecule according to the invention, an expression cassette according to the invention or an expression vector according to the invention is integrated in stable manner in the genome of the host cell.
For industrial applications, it would be ideal if the microorganism used were capable of metabolising all of the sugars present in the medium. Since the yeasts that are currently used are not capable of metabolising the arabinose in the medium, it would be highly advantageous to provide the strains with this additional capablity in stable manner. In order to achieve this, a nucleic acid molecule according to the invention, an expression cassette according to the invention or an expression vector according to the invention can be genomically integrated in stable manner and can allow for the metabolisation of arabinose in industrial strains. Using the nucleic acid molecules according to the invention ensures a very efficient arabinose conversion in industrial strains. Previously, the practice of introducing the genes of the bacterial L-arabinose metabolic pathway individually was associated with the difficulty that the genes were not present in an optimal ratio to each other. The transformations were time-consuming and the resulting arabinose metabolism was often not as efficient as desired. Moreover, the properties provided were often not stable. In contrast, the expression cassette according to the invention or the expression vector according to the invention, respectively, enable the bacterial L-arabinose metabolic pathway to be introduced quickly and functionally. With the selection of the promoters, it was possible to combine the genes together on one nucleic acid molecule, one expression cassette or one expression vector. The integration of the nucleic acid molecule according to the invention, the expression cassette according to the invention or the expression vector according to the invention, respectively, further guarantees an efficient arabinose conversion.
A host cell according to the invention is preferably a fungus cell, and more preferably a yeast cell, such as Saccharomyces species, Kluyveromyces sp., Hansenula sp., Pichia sp. or Yarrowia sp.
In particular, a host cell according to the invention is selected from BWY1, CEN.PK113-7D, Red Star Ethanol Red and Fermiol.
The object is further solved according to the invention by providing methods for producing bioethanol. One method according to the invention comprises the expression of a nucleic acid molecule according to the invention, an expression cassette according to the invention, or an expression vector according to the invention in a host cell.
Thereby, the method is preferably carried out in a host cell according to the invention.
The object is further solved according to the invention by the use of a nucleic acid molecule according to the invention, an expression cassette according to the invention, an expression vector according to the invention, or a host cell according to the invention to produce bioethanol.
The object is further solved according to the invention by the use of nucleic acid molecule according to the invention, an expression cassette according to the invention, an expression vector according to the invention, or a host cell according to the invention for recombinant fermentation of pentose-containing biomaterial.
For the methods and uses, see the examples and figures. The results of fermentation recorded in example 2 show that especially the codon-optimised genes of araA, araB and araD enable the yeast transform ants to metabolise arabinose more efficiently. The result of this is faster conversion of the sugar and a significantly higher ethanol yield.
The object is further solved according to the invention by providing a polypeptide selected from the group of
a. a polypeptide which is at least 70%, preferably at least 80% identical to the amino acid sequence that is coded by SEQ ID NO: 3, 4 or 5, and has an in vitro and/or in vivo pentose isomerase function,
b. a naturally occurring variant of a polypeptide including the amino acid sequence that is coded by SEQ ID NO: 3, 4 or 5, which has an in vitro and/or in vivo pentose isomerase function,
c. a polypeptide which is identical to the amino acid sequence that is coded by SEQ ID NO: 3, 4 or 5, and has an in vitro and/or in vivo pentose isomerase function, and
d. a fragment of the polypeptide from a., b. or c., comprising a fragment of at least 100, 200 or 300 continuous amino acids of the amino acid sequence that is coded by SEQ ID NO: 3, 4 or 5.
Such a polypeptide is preferably selected from the group of
a. a polypeptide which is at least 70%, preferably at least 80% identical to the amino acid sequence according to SEQ ID NO: 6 or 7, and has an in vitro and/or in vivo pentose isomerase function,
b. a naturally occurring variant of a polypeptide comprising the amino acid sequence according to SEQ ID NO: 6 or 7, which has an in vitro and/or in viva pentose isomerase function,
c. a polypeptide which is identical to the amino acid sequence according to SEQ ID NO: 6 or 7, and has an in vitro and/or in vivo pentose isomerase function, and
d. a fragment of the polypeptide from a., b. or c., comprising a fragment of at least 100, 200 or 300 continuous amino acids according to SEQ ID NO: 6 or 7.
A polypeptide according to the invention preferably comprises a polypeptide which is at least 90%, preferably 95% identical to the amino acid sequence that is coded by SEQ ID NO: 3, 4 or 5, and has an in vitro and/or in vivo pentose isomerase function.
Such a polypeptide according to the invention preferably comprises a polypeptide which is at least 90%, preferably 95% identical to the amino acid sequence according to SEQ ID NO: 6 or 7, and has an in vitro and/or in vivo pentose isomerase function.
The amino acid sequence with SEQ ID NO. 6 is the amino acid sequence of Bacillus licheniformis L-arabinose isomerase (araA). This amino acid sequence is preferably coded by the nucleic acid sequences with SEQ ID NOs. 3 or 4.
The amino acid sequence with SEQ ID NO. 7 is the amino acid sequence of Clostridium acetobutylicum L-arabinose isomerase (araA). This amino acid sequence is preferably coded by the nucleic acid sequence with SEQ ID NO. 5.
The pentose is arabinose, in particular L-arabinose.
The polypeptide according to the invention preferably originates from a bacterium, more preferably from Bacillus licheniformis or Clostridium acetobutylicum. 
These L-arabinose isomerases are advantageous for the growth of yeast transformants on arabinose medium. A number of different experiments indicated that the L-arabinose isomerase from B. subtilis that was used previously represents a limiting step in the decomposition of arabinose in yeast (Becker and Boles, 2003; Wiedemann, 2003; Karhumaa et al, 2006; Sedlak and Ho, 2001). Example 1 shows (see also FIG. 4) that the growth of yeast transformants on arabinose medium is significantly improved particularly by the expression of L-arabinose isomerase from C. acetobutylicum and from B. licheniformis, in comparison to the isomerase from B. subtilis. 
The object is further solved according to the invention by providing an isolated nucleic acid molecule that codes for a polypeptide according to the invention.
Additionally, the object is further solved according to the invention by providing a host cell that contains such an isolated nucleic acid molecule.
For preferred embodiments of the isolated nucleic acid molecule and of the host cells, reference is made to the embodiments described above.
The polypeptide according to the invention, the isolated nucleic acid molecule according to the invention and the host cell according to the invention are preferably used in the production of bioethanol and for recombinant fermentation of pentose-containing biomaterial.
A further aspect of the present invention are host cells that contain one or more modifications, such as nucleic acid molecules.
An additional modification of such kind is a host cell that overexpresses a TAL1 (transaldolase) gene, such as is described by the inventors in EP 1 499 708 B1, for example.
A further such additional modification is a host cell that contains a nucleic acid coding for a specific L-arabinose transporter gene (araT), particularly such as a specific L-arabinose transporter gene from the genome of P. stipitis, such as is described by the inventors in German Patent Application DE 10 1006 060 381.8, filed on Dec. 20, 2006.
Further biomass with significant amounts of arabinose (source of the data: U.S. Department of Energy:
Type of biomassL-arabinose [%]Switchgrass3.66Large bothriochloa3.55Tall fescue3.19Robinia3Corn stover2.69Wheat straw2.35Sugar cane bagasse2.06Chinese lespedeza1.75Sorghum bicolor1.65
The nucleic acids, expression cassettes, expression vectors and host cells according to the invention are also of great importance for their utilization.
Possible uses of the nucleic acids, expression cassettes, expression vectors and host cells according to the invention include both the production of bioethanol and the manufacture of high-quality precursor products for further chemical synthesis.
The following list originates from the study “Top Value Added Chemicals From Biomass”. Here, 30 chemicals, which can be produced from biomass, were categorized as being particularly valuable.
Number ofC atomsTop 30 Candidates1hydrogen, carbon monoxide23glycerol, 3-hydroxypropionic acid, lactic acid,malonic acid, propionic acid, serine4acetoin, asparaginic acid, fumaric acid, 3-hydroxy-butyrolactone, malic acid, succinic acid, threonine5arabitol, furfural, glutamic acid, itaconic acid,levulinic acid, proline, xylitol, xylonic acid6aconitic acid, citrate, 2,5-furandicarboxylic acid,glucaric acid, lysine, levoglucosan, sorbitol
It is important to have the nucleic acids, expression cassettes, expression vectors and host cells according to the invention available as soon as these chemicals are produced from lignocellulose by biokonversion (e.g. fermentations with yeasts).
FIG. 1 Composition of the biomass.
Biomass consists of cellulose, hemicellulose and lignin. The second most frequently occurring hemicellulose is a highly branched polymer consisting of pentoses, uronic acids and hexoses. The hemicellulose consists in a large proportion of the pentoses xylose and arabinose.
FIG. 2 Scheme of the metabolism of L-arabinose in recombinant S. cerevisiae by integration of a bacterial L-arabinose metabolic pathway.
FIG. 3 Vectors used and their construction.
The initial plasmid for construction of the vector p425H7synthAra (FIG. 3 A) was the plasmid p425HXT7-6HIS (FIG. 3 B). The open reading frames of the codon-optimised genes of araA from B. licheniformis and araBmut and araD from E. coli were amplified and cloned into the plasmid p425HXT7-6HIS after various promoters and terminators. The primers were selected in such manner that the resulting expression cassette was flanked by the restriction sites of enzymes PacI and AscI. Thereby, the plasmid p425H7synthAra was produced, which has a leucine marker.
FIG. 4 Growth on arabinose using various L-arabinose isomerase genes.
Growth curves of recombinant S. cerevisiae strains containing the bacterial L-arabinose metabolism with various L-arabinose isomerases. Growth tests were conducted in 5 ml SM medium with 2% arabinose under aerobic conditions. The L-arabinose isomerases of C. acetobutylicum, B. licheniformis, P. pentosaceus, L. plantarum and L. mesenteroides were tested. The L-arabinose isomerase from B. subtilis and the empty vector p423HXT7-6HIS were used as controls.
FIG. 5 Growth on arabinose using codon-optimised arabinose metabolic pathway genes.
Growth curves of recombinant S. cerevisiae strains containing the bacterial L-arabinose metabolism with different combinations of codon-optimised genes and the genes with original sequences. Growth tests were conducted in 5 ml SM medium with 2% arabinose under aerobic conditions. Each of the combinations that contained one of the codon optimised genes respectively, and the combination containing all three codon-optimised genes were tested. In addition, the combination in which the codon-optimised genes of kinase and epimerase were present was also tested. A recombinant yeast strain with the four genes having the original sequences was used as a control.
FIG. 6 Ethanol formation using codon-optimised arabinose metabolic pathway genes.
The figure shows the results of HPLC analyses of the media supernatants from two fermentations. One fermentation was carried out with strain BWY1, which possesses plasmids p423H7synthIso, p424H7synthKin, p425H7synthEpi and pHL125re (3× synth). In the other fermentation, strain BWY1 was tested, containing plasmids p423H7araABsre, p424H7araBre, p425H7araDre and pHL125re (3×re). The fermentations were carried out in SFM medium with 3% L-arabinose. The strains were grown to a high optical density in the fermenter. Then, the fermentation was changed to anaerobic conditions (after 48 hours). The plots show arabinose consumption and ethanol production.
FIG. 7 Growth on arabinose using the constructed expression plasmid p425H7-synthAra.
Growth curves of recombinant S. cerevisiae strains containing bacterial L-arabinose metabolism in the form of the vector p425H7-synthAra. Growth tests were conducted in 5 ml SC medium with 2% arabinose under aerobic conditions. A recombinant yeast strain with the plasmids p423H7araABsre, p424H7araBre, p425H7araDre and pHL125re, which had been tested in 5 ml SM medium with 2% arabinose, was used as the control.